Spectroscopy 14 (1998) 35–40 35 IOS Press Total assignment of the 1 H and 13 C NMR spectra of piperovatine Willy Rendón a , Galia Chávez a , Myriam Meléndez-Rodríguez b and Pedro Joseph-Nathan b,* a Instituto de Investigaciones Químicas, Universidad Mayor de San Andrés, La Paz, Bolivia b Departamento de Química, Centro de Investigación y de Estudios Avanzados, Instituto Politécnico Nacional, Apartado 14-740, México, D.F., 07000 Mexico Abstract. Total and unambiguous assignment of the 1 H NMR spectrum of piperovatine [6-(4-methoxyphenyl)-N -(2-methyl- propyl)-2,4-hexadienamide] was carried out using conventional 1D NMR methods and spectral spin–spin simulation. Based on these data, the complete assignment of the 13 C NMR chemical shift values was made by a 13 C/ 1 H chemical shift correlation diagram and conventional considerations for the quaternary carbon atom. 1. Introduction Piperovatine (1) [6-(4-methoxyphenyl)-N -(2-methylpropyl)-2,4-hexadienamide] is a naturally occur- ring alkaloid, which exhibits insecticidal [1] and local anesthetic activity [2,3]. It has been isolated mainly from several Ottonia [3–5] and Piper species [2,6]. Although syntheses of the alkaloid have been carried out [4,7–9], detailed NMR investigations of this interesting natural product have, to our knowledge, not been performed. The 1 H NMR spectrum of 1 has previously been reported [4,8] but the assignment of the spectral data remained ambiguous; besides, a 13 C NMR study has not been described. In this work we wish to report the unambiguous and complete assignment of the 1 H and 13 C NMR spectra of 1, isolated during a phytochemical study of Piper darienence, which is used in the bolivian folk medicine against toothache. * Corresponding author. 0712-4813/98/$8.00 1998 – IOS Press. All rights reserved
Transcript
Spectroscopy 14 (1998) 35–40 35IOS Press
Total assignment of the1H and13C NMRspectra of piperovatine
Willy Rendóna, Galia Cháveza, Myriam Meléndez-Rodríguezb and Pedro Joseph-Nathanb,∗
a Instituto de Investigaciones Químicas, Universidad Mayor de San Andrés, La Paz, Boliviab Departamento de Química, Centro de Investigación y de Estudios Avanzados, Instituto PolitécnicoNacional, Apartado 14-740, México, D.F., 07000 Mexico
Abstract. Total and unambiguous assignment of the1H NMR spectrum of piperovatine [6-(4-methoxyphenyl)-N-(2-methyl-propyl)-2,4-hexadienamide] was carried out using conventional 1D NMR methods and spectral spin–spin simulation. Based onthese data, the complete assignment of the13C NMR chemical shift values was made by a13C/1H chemical shift correlationdiagram and conventional considerations for the quaternary carbon atom.
1. Introduction
Piperovatine (1) [6-(4-methoxyphenyl)-N -(2-methylpropyl)-2,4-hexadienamide] is a naturally occur-ring alkaloid, which exhibits insecticidal [1] and local anesthetic activity [2,3]. It has been isolated mainlyfrom severalOttonia[3–5] andPiperspecies [2,6]. Although syntheses of the alkaloid have been carriedout [4,7–9], detailed NMR investigations of this interesting natural product have, to our knowledge, notbeen performed. The1H NMR spectrum of1 has previously been reported [4,8] but the assignment of thespectral data remained ambiguous; besides, a13C NMR study has not been described. In this work wewish to report the unambiguous and complete assignment of the1H and13C NMR spectra of1, isolatedduring a phytochemical study ofPiper darienence, which is used in the bolivian folk medicine againsttoothache.
* Corresponding author.
0712-4813/98/$8.00 1998 – IOS Press. All rights reserved
36 W. Rendón et al. / Total assignment of the1H and13C NMR spectra
2. Experimental
2.1. Plant material
Piper darienenceD.C. was collected in the neighborhood of the Blanco river, between Remancitoand Cafetal, in the Beni department, Itenez province, Bolivia, in February 1996. It was classified by Dr.Stephan Beck from the National Herbarium of Bolivia, where a sample (voucher no. 4012) is deposited.
2.2. Extraction and isolation
Air dried roots (532 g) ofPiper darienenceD.C. were extracted with ethanol after removing thegrease with petroleum ether. The ethanol was evaporated under vacuum and the residue was washed withCHCl3. The chloroform was evaporated and the residue chromatographed by column chromatography(CC) on silica gel 60 Å (Aldrich, 70–230 mesh). A fraction of 500 mL was collected using chloroformand then 8 fractions of 300 mL were collected with ethanol. The second ethanolic fraction was purified byTLC on silica gel (Fluka A.G., Buchs SG kieselgel GF254) using chloroform–ethyl acetate (10 : 2 v/v).Compound1 was obtained as a solid, which was crystallized from ethyl acetate (300 mg). It was furtherpurified on column chromatography using silica gel 60 Å (Merck, 230–400 mesh) and chloroform–ethyl acetate (10 : 2 v/v). Crystallization from ethanol gave colorless needles, m.p. 121–122◦. Theidentity of 1 was confirmed by comparing its physical and NMR spectral data with those reported in theliterature [4].
2.3. Nuclear magnetic resonance instrumental conditions
The 1H and 13C NMR spectra were recorded at 300 and 75.4 MHz, respectively, on a Varian XL-300GS spectrometer using CDCl3 with TMS as the internal reference. Measurements were performedat ambient probe temperature using 5 mm o.d. sample tubes. For the13C/1H chemical shift correlationexperiment, the standard pulse sequence was used [10,11]. The spectra were acquired with 1024 datapoints and 128 time increments with 128 transients per increment. Thef1 andf2 spectral widths were12048.2 and 2334.8 Hz, respectively. The relaxation delay was 1 s and an average1J(C,H) was set to140 Hz.
3. Results and discussion
In previous1H NMR spectral reports of1 [4,8] H-2′′, H-3, H-4 and H-5 are mentioned only as multi-plets and the signals for the aromatic H-2′, H-3′, H-5′ and H-6′ are not assigned at all. Using conventional1D NMR methods and spectral spin–spin simulation we describe herein the precise chemical shift andcoupling constant values for each of these protons, as is shown in Table 1.
Inspection of the1H NMR spectrum of1 showed at first glance the H-2′′ signal as a septet. However,such a multiplicity is discarded because the intensity ratio between the higher intensity signals for a septetis 20/15 = 1.33 in contrast with the spectrometer digitally readed signal intensities 135.485/107.925 =1.255, which clearly corresponds to a nonet (70/56 = 1.25). An amplitude increased plot shows that theH-2′′ signal is found as a nonet (J = 6.7 Hz) centered at 1.79 ppm with intensity ratio 1 : 8 : 28 : 56 :70 : 56 : 28 : 8 : 1, as expected for an isobutyl methine proton. A selective irradiation of this signalsimplifies, from a double doublet to a doublet, the signal at 3.16 ppm assigned to the protons of the
W. Rendón et al. / Total assignment of the1H and13C NMR spectra 37
Table 11H and13C NMR spectral assignments of piperovatine (1)
4′ 158.11′′ 3.16 dd 6.7, 6.1 46.92′′ 1.79 nonet 6.7 28.63′′ 0.92 d 6.7 20.14′′ 0.92 d 6.7 20.1
MeO 3.79 s 55.2NH 5.49 brs
Fig. 1. (a) Section of the1H NMR spectrum and (b) spin–spin simulation of piperovatine (1).
methylene group (H-1′′) and from a doublet to a singlet, the signal at 0.92 ppm assigned to the protonsof the methyl groups (H-3′′ and H-4′′).
The H-3 signal is shown at 7.21 ppm as a double doublet (J = 14.9, 10.0 Hz) through couplings withH-2 and H-4. These couplings were confirmed by selective irradiation.
38 W. Rendón et al. / Total assignment of the1H and13C NMR spectra
Fig. 2. Heteronuclear13C/1H chemical shift correlation diagram of piperovatine (1).
W. Rendón et al. / Total assignment of the1H and13C NMR spectra 39
The frequencies for H-4 and H-5 appear superimposed in the range 6.08–6.25 ppm. The assignmentof their individual resonance frequencies and multiplicities was achieved through selective homonuclearirradiations combined with spectral spin–spin simulation. In agreement with an inspection of1, and con-sidering only vicinal couplings, the multiplicities for H-4 and H-5 must correspond to a double doubletand to a double triplet, respectively. Thus, irradiation of the signal at 7.21 ppm (H-3) simplified thedouble doublet signal at 6.12 ppm for H-4 to a doublet. On the other hand, irradiation of the signal at3.42 ppm (H-6) simplified the double triplet signal at 6.20 ppm for H-5 to a doublet. In order to performthe precise assignment of the H-4 and H-5 signals, a spectral spin–spin simulation [12] was made. There-fore a six spins case for the H-2, H-3, H-4, H-5 and 2 H-6 nuclei was solved iteratively. The results fittedsatisfactorily with the experimental data (RMS error frequency 0.13), as shown in Fig. 1.
The assignment of the aromatic H-2′, H-3′, H-5′ and H-6′ signals (AA′BB′ pattern) was made on thebasis of their chemical shift [13]. Thus the signal at 7.08 ppm was identified as belonging to H-2′ andH-6′, while the signal at 6.84 ppm was assigned to H-3′ and H-5′.
The remaining proton resonance assignments for H-1′′, H-3′′, H-4′′ and the NH group were confirmedby selective proton irradiations and the interchange with deuterium oxide.
The six proton doublet signal (J = 6.7 Hz) at 0.92 ppm assigned to H-3′′ and H-4′′ was simplifiedto a singlet by irradiation of the signal at 1.79 ppm (H-2′′). The two proton double doublet signal (J =6.7, 6.1 Hz) at 3.16 ppm assigned to H-1′′ was simplified to a doublet by irradiation of the signal at1.79 ppm (H-2′′) and, on addition of deuterium oxide, it also collapsed to a doublet. The broad singlet at5.49 ppm assigned to the NH group interchanged with deuterium oxide. The complete1H NMR data of1 are given in Table 1.
Once the complete1H NMR spectrum of1 had been assigned, a13C/1H chemical shift correlation ex-periment allowed the assignment of all protonated carbons (Fig. 2). The non-protonated C-1 carbonyl sig-nal appears at 166.2 ppm and the aromatic carbons C-1′ and C-4′ were assigned at 131.1 and 158.1 ppm,respectively, by application of additivity relationships [13]. The13C NMR chemical shift values are sum-marized in Table 1.
Acknowledgements
We are grateful to Isaias Chávez S., Pedro Arza and Luchi Muñoz for etnomedical information andplant collection, to Dr. Stephan Beck from National Herbarium of Bolivia for the botanic classificationand to CYTED-Spain for stimulating support.
References
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